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Int.J.Curr.Microbiol.App.Sci (2019) 8(2): 3350-3362 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 02 (2019) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2019.802.390 Tapping of Root Non-Rhizobial Endophytic Bacteria from Chickpea Plant Tissues for Multifunctional Traits Deepika Chhabra1* and Poonam Sharma2 1 Department of Microbiology, Punjab Agricultural University, Ludhiana-114004, Punjab, India 2 Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana-114004, Punjab, India *Corresponding author ABSTRACT Keywords Chickpea, Endophytes, Non rhizobial Article Info Accepted: 22 January 2019 Available Online: 10 February 2019 In the present investigation total 167 non rhizobial endophytic bacterial isolates from roots of chickpea were collected and screened for qualitative P solubilization. Out of these 37 potential bacterial endophytic isolates from root were selected for further quantitative Psolubilization, Indole acetic acid (IAA), Gibberelic acid and ACC deaminase production. High P-solubilization was recorded in isolate RBR20 (20.60 mg100ml1). Maximum amount of IAA was produced by isolate LCRE 9 (39.60 μgml-1) (presence of tryptophan) and RBR164 (19.93 μgml-1) (absence of tryptophan). High amount of GA production was observed in RBR19, RBR127, RBR136 and RBR164 (112.15 μgml -1). RBR164 isolate showed highest growth in DF medium with ACC (0.9985). There is need to exploit non rhizobial endophytic bacteria with plant growth promoting traits (PGP) as single or consortium biofertilizer for sustainable agriculture. Introduction Non rhizobial endophytic bacteria were characterized from different plant species in last couple of years have raised their prospects to be used as biofertilizer (Akhtar and Siddiqui 2009). Endophytic bacteria are defined as interior colonizers of the plant viz. root, seed, stem and leaf without showing any harmful impact on host plant. Endophytic microorganism can promote plant growth, accelerate seed emergence with enhanced plant establishment under stressful conditions in legume and non-legume plants to amass nutrients by different processes viz. phosphate solubilization (Wakelin et al., 2004), iron chelation (Ryan 2008), preventing disease via antifungal and outcompeting pathogens for nutrients with siderophore production and better plant general resistance. Endophytic plant growth promoting bacteria produces phytohormones, volatile compounds and co factors such as pyrroquinoline quinine (PQQ), that stimulate growth of plant). Endophytic bacteria directly contribute to the growth of host plant by growth regulator production like auxins, gibberellins and cytokinins (Bhattacharyya and Jha 2012). Several plant associated bacteria are shown to 3350 Int.J.Curr.Microbiol.App.Sci (2019) 8(2): 3350-3362 supply auxins, indole-3-acetic acid (IAA), which boosts lateral root formation and therefore, nutrient uptake and root exudation by plants. Endophytic bacteria with P solubilization activity (PSBs) embrace Bacillus megaterium, B. circulans, B. subtilis, genus Pseudomonas straita, P. rathonis; chiefly attributed due to production of organic acids like carboxylic acid, ethanedioic acid, glyoxalic acid, malic acid, hydroxy acid and ketobutyric acid (Reyes et al., 2007). In recent decades, interest in endophytic microorganisms has been increased, as they have important role in sustainable agriculture. Knowing and understanding the negative impact of artificial fertilizers in agriculture, novel approaches such as the application of endophytic bacteria as biofertilizer which are associated with plants, may help to increase productivity and improve plant health. Materials and Methods Plant Growth Promotional (PGP) traits for isolates of endophytic bacteria Determination solubilization of Phosphate Quantitative estimation Pikovaskaya’s broth (100 ml) and 0.1g P2O5 as tri-calcium phosphate (TCP) was added in 250 ml conical flask as an inorganic phosphate substrate and the flasks containing broth were autoclaved at 121° C for 15 min. The broth was inoculated with 1 ml of overnight grown pure culture suspension and incubation was done at 28±2°C for 15 days. Equal ratio of ammonium molybdate and ammonium vandate was added to culture supernatant and incubated for 25 minutes, development of yellow colour indicated phosphate solubilizing activity. Intensity of the yellow colour of solution was measured spectroscopically (Elico UV-VIS spectrophotometer) at 420 nm for quantitative estimation (Jackson, 1973). Qualitative and quantitative analysis for Indole acetic acid (IAA) production (P) Phosphate solubilization index (PSI) “Qualitative assay for P solubilization of plant associated endophytic bacteria was done by streaking of pure culture on NBRIP medium containg plates (National Botanical Research Institute’s Phosphate growth) (Arora 2007). “ Appearence of clear halo zone around bacterial colony after 5-7 days incubation period at 28±2oC was indicated positive for P solubilization (Nautiyal 1999). Following formula was used to calculate Phosphate solubilization index (PSI): PSI Index= A/B A= Total diameter (colony + halo zone) B= Diameter of colony. Further quantitative phosphate solubilization was performed with promising P solubilizers with presence of clear halo zone around bacterial growth in plate assay method. IAA production in different isolates of endophytes were detected (Gordon and Weber 1951) by inoculating pure bacterial culture in 10 ml Luria Bertanni broth with or without tryptophan (0.01% L-Trp) and incubation was done at 28-30°C for 3-6 days. Presence of pink colour showed production of IAA which was indicative of positive test. Quantative estimation was done for IAA (µgml-1) by addition of 2 ml of Salkowski’s reagent (1 ml of 0.5m FeCl3 in 50 ml of 35% HClO4) into culture supernatant (1 ml) alongwith uninoculated broth with Salkowski’s reagent as a reference. After 20 min, absorbance of pink colour was measured spectroscopically (Elico UV-VIS spectrophotometer) at 535 nm and quantification of IAA was done by using standard curve. 3351 Int.J.Curr.Microbiol.App.Sci (2019) 8(2): 3350-3362 Quantative measurement of Gibberellic acid production Quantative measurement of gibberellic acid by endophytic bacteria was estimated as per method of Borrow et al., (1995). with ACC as a sole nitrogen source were streaked with pure culture of endophytic isolates and bacterial growth was observed (Dworkin and Foster, 1958). Incubation was done for 3-4 days at 28±1°C. Quantitative estimation Reagents Zinc acetate (21.9g) was added into 80ml of distilled water and 1ml of glacial acetic acid to make the volume upto 100ml with distilled water. Liquid DF minimal medium with (NH4)2SO4, with ACC (sigma, Ltd) and without ACC was used to culture endophytic isolates individually. Growth of bacterial isolates in different media was measured at 600nm by using UV-VIS Spectrophotometer (Shahzad et al., 2010). Potassium ferrocyanide solution Results and Discussion Potassium ferrocyanide (10.6g) was mixed in 100ml distilled water. Cultures inoculated in their relevant broth containing tubes and incubation was done at 370C for seven days. After end of incubation period, cultures were centrifuged for 10 min at 8000 rpm. After centrifugation two ml of zinc acetate solution was added into fifteen ml of the culture supernatant. After two minutes, 2 ml of potassium ferrocyanide solution was added and again centrifuged for 10 min at 8000 rpm. Equal volume of supernatant (5 ml) was added to 30 % hydrochloric acid (5 ml) and the test tube was incubated at 27°C for 1 hr 15min. HCL (5%) was used as a blank. UV-VIS spectrophotometer was used to measure the absorbance at 254nm. Gibberellic acid solution of known strength was used to prepare standard curve to quantify the gibberellic acid produced by the cultures and expressed as μgml-1 broth. Phosphorus is the key element in the nutrition of plants, next to nitrogen (N). It plays an important role in virtually all major metabolic processes in plant including photosynthesis, energy transfer, signal transduction, macromolecular biosynthesis, respiration and nitrogen fixation in legumes. For qualitative P- solubilization, all the 167 isolates were tested on NBRIP medium amended with 0.5% tri calcium phosphate (TCP) as inorganic source of phosphorus. 37 isolates from root were positive for P- solubilization on NBRIP medium. Phosphate solubilization index (PSI) of endophytic bacterial isolates was ranged from 1.25 to 2.22. Out of 37 roots non rhizobial endophytic isolates 32 % were shown high PSI from root. Highest P solubilization index was recorded with RBR 164 (2.22) followed by LCRE9 (2.15) isolates. Our results are in accordance with Liu et al., (2017) who reported out of 28 isolates of endophytic bacteria from tomato rhizosphere, 24 were able to form halo yellow zone on Pikovaskaya’s and NBRIP medium. Kailasan and Vamanrao (2015) reported 23 isolates of 134 showed solubilization index ≥ 1. PSB isolates showed maximum amount of Psolubilization at 12th days and few at15th day Zinc acetate solution Determination production of ACC deaminase Qualitative assay was done as per method of Govindasamy et al., (2008). Plates containing Dworkin’s and Foster (DF) minimal medium 3352 Int.J.Curr.Microbiol.App.Sci (2019) 8(2): 3350-3362 and varied from 2.70 to 20.60 mg 100ml-1. Significantly high P-solubilization was recorded in root isolate RBR20 (20.60 mg100ml-1) followed by RBR164 (19.80 mg 100ml-1) of chickpea (Fig. 1 and Table 1). Production of phytohormone (IAA) is an important mechanism of plant growth promotion by endophytic bacteria. This hormone promotes the growth of roots. Of 37 root endophytic bacterial isolates 59.37%, 8.10% and 32.43% were found to be low, medium and high producer in the presence of tryptophan (Fig. 2). IAA production in root non rhizobial endophytic bacterial isolates ranged from 4.50- 19.93 μgml-1 (absence of tryptophan) and 21.0-39.60 μgml-1(presence of tryptophan). In the presence of tryptophan, the isolate LCRE 9 produced the maximum amount of IAA (39.60 μgml-1) whereas in the absence of tryptophan the isolate RBR164 produced the maximum amount of IAA (19.93 μgml-1) as given in Table 2. Our results are in close agreement with Priyanka and Leelawati (2015) where of 8 non rhizobial endophytic bacteria from chickpea nodules. In 4 isolates IAA varied from 6.33 µg ml-1 to10.04 µg ml1 in the presence of tryptophan. Our results are also in harmony with the finding of Zaghloul et al., (2016) where of 55 non rhizobial endophytic bacterial isolates 12 produced ˃ 25 IAA in the presence of tryptophan with maximum amount by the isolate RN62 (92.52 µ/ml) Endophytic bacteria have many beneficial effects on their host plant growth by producing phytohormones similar to that of plant growth promoting rhizobacteria (PGPR). Gibberellic acid (GA) is an important plant growth promoter associated with several plant growth and development processes, such as seed germination, stem elongation, flowering and fruit development. Of 37 root non rhizobial endophytic bacterial isolates 16.21%, 51.35% and 32.43% and 38 from were found to be low, medium and high producer of GA, respectively (Fig. 3). Twelve endophytic bacterial isolates viz. RBR17, RBR 19, RBR20, RBR 40, RBR 127, RBR 136, RBR 146, RBR 155, RBR 164, RBR 167, LCRE 8 and LCRE 9 were tended to produce high amount of GA in the range of 59.90 to 112.15 μgml-1. High amount of GA production was observed in RBR19, RBR127, RBR136, and RBR164 (112.15 μgml-1) procured from wild species of chickpea. Our results are well coherent with Asaf et al., (2017) who isolated 5 bacterial endophytes LK11 (Sphingomonas sp. LK11), TP5 (Bacillus subtilis), MPB5.3 (B. subtilis subsp. Subtilis), S9 (B. subtilis subsp. Subtilis), and TP1 (Serratia marcescens) from arid land-dwelling plants. Enhancement in soybean plants (155.43–146.94 ng/g D.W.) was recorded with gibberellin production endophyte-inoculated as compared to control (113.76 ng/g D.W.). All 37 endophytic bacterial isolates were subjected for their ACC deaminase production on Dworkin and Foster’s minimal medium. The ability of isolates to utilize ACC as a source of N was assessed on the basis of bacterial growth on plates containing substrate ACC and (NH4)2SO4. Based on preliminary qualitative test for screening of endophytic bacterial isolates positive for ACC deaminase, 14 isolates were further assessed on the basis of bacterial growth in liquid medium selected for their ability to utilize ACC as a sole source of N in terms of optical density (OD at 600). Higher growth of endophytic bacterial isolates was observed in DF broth supplemented with ACC (OD ranged 0.356 to 0.9985) as compared to DF broth supplemented with (NH4)2 SO4 (OD ranged 0.2954 to 0.5932) (Table 6). Data indicated the ability of isolates to use ACC as N source due to the presence of ACC deaminase activity. Little or no growth in DF medium without ACC or (NH4)2 SO4 was observed due to the absence of N source. RBR164 isolate showed highest growth in DF medium with ACC (0.9985) followed by RBR83 (0.9935) (Table 3–6) (Fig. 4). 3353 Int.J.Curr.Microbiol.App.Sci (2019) 8(2): 3350-3362 Table.1 Qualitative measurement of P-solubilization by root non rhizobial endophytic bacterial isolates of chickpea on NBRIP medium Isolates RBR11 RBR14 RBR17 RBR 19 RBR20 RBR25 RBR27 RBR31 RBR34 RBR38 RBR40 RBR49 RBR57 RBR61 RBR63 RBR72 RBR75 RBR80 RBR83 RBR89 RBR112 RBR116 RBR119 RBR121 RBR127 RBR128 RBR136 RBR139 RBR144 RBR145 RBR146 RBR155 RBR164 RBR165 RBR167 LCRE8 LCRE9 LGR33 RB1 Colony Diameter 0.9 1.1 1.1 1.1 1 1 1.2 1.3 1 0.8 1 0.9 0.8 0.9 0.9 1 1.2 1.3 1 1 1 1.1 0.8 0.7 0.8 1.1 1 1.2 1.2 0.9 1 1.1 0.9 1.1 0.9 1.1 1.3 1.2 0.7 Colony + Holo Zone Diameter 1.5 1.8 2.1 2 2.1 1.6 2.1 2.2 1.7 1.4 2 1.6 1 1.6 1.5 1.7 1.6 1.7 1.5 1.6 1.5 1.8 1.3 1.1 1.5 1.9 2.0 2 1.6 1.6 2 2.1 2 1.7 1.9 2.1 2.8 1.9 1.3 3354 PSI 1.67 1.64 1.91 1.82 2.10 1.60 1.75 1.69 1.70 1.75 2.00 1.78 1.25 1.78 1.67 1.70 1.33 1.31 1.50 1.60 1.50 1.64 1.63 1.57 1.87 1.73 2.0 1.67 1.33 1.78 2.00 1.91 2.22 1.55 2.11 1.91 2.15 1.58 1.86 Int.J.Curr.Microbiol.App.Sci (2019) 8(2): 3350-3362 Table.2 Quantitative measurement of P-solubilization by root non rhizobial endophytic bacterial isolates in Pikovaskaya’s broth at a different interval of time Isolates RBR11 RBR14 RBR17 RBR 19 RBR20 RBR25 RBR27 RBR31 RBR34 RBR38 RBR40 RBR49 RBR57 RBR61 RBR63 RBR72 RBR75 RBR80 RBR83 RBR89 RBR112 RBR116 RBR119 RBR121 RBR127 RBR128 RBR136 RBR139 RBR144 RBR145 RBR146 RBR155 RBR164 RBR165 RBR167 LCRE8 LCRE9 LGR33 RB1 CD @ 5% 3rd 1.1 2.2 1.0 1.3 8.9 0.3 3.0 0.3 0.5 4.5 1.2 3.7 0.3 0.3 6.3 0.3 4.0 1.0 1.6 2.9 4.6 0.4 1.6 1.7 0.3 4.4 4.8 4.3 0.3 3.0 9.5 3.5 5.3 4.9 5.0 6.57 2.39 2.6 4.5 0.35 P-solubilization (mg100ml-1) Incubation period (days) 6th 9th 12th 2.5 4.4 4.5 4.8 5.1 6.1 2.2 15.3 16.8 2.9 5.3 17.6 19.3 19.9 20.6 1.0 4.8 4.9 4.4 10.1 11.1 2.6 4.4 6.0 5.9 8.4 10.5 6.0 11.0 12.1 7.4 8.8 12.3 5.1 13.2 13.4 2.6 2.4 2.7 1.4 6.2 8.1 9.4 9.6 10.1 5.7 5.7 7.7 4.4 6.7 9.9 5.2 5.9 10.8 3.1 10.0 10.7 4.8 10.0 10.7 7.4 10.3 11.3 1.4 4.4 10.6 1.7 2.9 3.8 4.8 4.9 5.8 2.1 5.4 12.3 5.1 5.7 10.6 4.9 13.8 15.8 5.0 8.6 10.9 2.2 2.4 4.5 4.5 5.5 6.2 13.3 14.7 15.4 5.0 12.7 9.0 5.6 16.9 19.8 7.2 8.1 8.8 6.4 13.0 13.6 14.19 15.69 16.07 4.42 9.76 12.86 4.1 4.9 8.7 7.32 7.31 10.19 0.39 0.15 0.39 3355 15th 5.2 5.76 7.8 6.4 18.0 3.9 12.8 5.7 2.8 4.7 6.9 8.3 2.6 9.8 6.4 6.5 3.3 5.1 6.2 5.5 5.6 3.1 7.3 2.1 6.9 4.9 5.0 8.8 6.9 5.0 8.0 6.4 7.5 3.7 17.5 15.44 3.86 3.4 7.75 0.45 Int.J.Curr.Microbiol.App.Sci (2019) 8(2): 3350-3362 Table.3 Quantitative measurement of IAA production by root non rhizobial endophytic bacterial isolates of chickpea Isolates RBR11 RBR14 RBR17 RBR 19 RBR20 RBR25 RBR27 RBR31 RBR34 RBR38 RBR40 RBR49 RBR57 RBR61 RBR63 RBR72 RBR75 RBR80 RBR83 RBR89 RBR112 RBR116 RBR119 RBR121 RBR127 RBR128 RBR136 RBR139 RBR144 RBR145 RBR146 RBR155 RBR164 RBR165 RBR167 LCRE8 LCRE9 LGR33 RB1 CD @ 5% Without Tryptophan (6th Day) (µ/ml) 7.98 8.17 12.51 12.23 17.83 8.14 9.19 9.70 9.38 9.98 14.70 7.57 5.08 7.71 8.21 7.39 5.40 4.50 7.42 9.07 8.82 7.88 9.37 7.60 14.70 8.01 11.98 9.38 9.35 6.79 14.13 14.30 19.93 10.37 13.27 14.28 16.10 15.28 14.23 0.02 3356 With Tryptophan (6th Day) (µg/ml) 21.70 21.37 30.75 31.35 32.81 24.10 21.00 21.71 22.71 22.64 34.93 23.24 27.08 21.62 24.92 24.50 23.71 23.17 22.04 21.35 21.25 21.10 21.43 22.82 32.84 22.90 33.21 22.61 24.08 27.66 31.68 39.00 39.31 21.80 37.20 37.16 39.60 32.45 35.72 0.08 Int.J.Curr.Microbiol.App.Sci (2019) 8(2): 3350-3362 Table.4 Quantitative estimation of gibberellic acid by root non rhizobial endophytic bacterial isolates of chickpea Isolates RBR11 RBR14 RBR17 RBR 19 RBR20 RBR25 RBR27 RBR31 RBR34 RBR38 RBR40 RBR49 RBR57 RBR61 RBR63 RBR72 RBR75 RBR80 RBR83 RBR89 RBR112 RBR116 RBR119 RBR121 RBR127 RBR128 RBR136 RBR139 RBR144 RBR145 RBR146 RBR155 RBR164 RBR165 RBR167 LCRE8 LCRE9 RB1 LGR33 CD @ 5% GA3 (μg ml-1) 85.39 96.63 101.56 112.15 111.38 95.48 89.62 95.25 91.76 91.28 108.53 100.29 92.15 94.15 83.73 97.15 98.13 86.00 92.15 96.83 93.60 59.90 92.15 96.01 112.15 92.33 112.15 94.21 95.33 99.59 101.83 108.45 112.15 92.15 108.64 104.64 105.51 80.65 111.91 0.41 3357 Int.J.Curr.Microbiol.App.Sci (2019) 8(2): 3350-3362 Table.5 Qualitative ACC deaminase production by non rhizobial endophytic Bacteria of chickpea Broth ACC deaminase positive non rhizobial endophytic bacterial isolates DF RBR12, RBR14,RBR61, RBR72, RBR89, RBR119, RBR121, RBR127, RBR 139, RBR 146 DF + (NH4)SO4 RBR12, RBR17, RBR19, RBR20, RBR34, RBR40, RBR72, RBR80, RBR83, RBR89, RBR119, RBR121, RBR127, RBR136, RBR139, RBR146, RBR155 & LCRE8 DF+ACC RBR12, RBR17, RBR19, RBR20, RBR25, RBR34, RBR40, RBR72, RBR80, RBR83, RBR89, RBR119, RBR121, RBR127, RBR136, RBR139, RBR144, RBR146, RBR155, RBR164, RBR165 & LCRE8 Table.6 Quantative ACC deaminase production by non rhizobial endophytic bacteria from chickpea in DF minimal medium Isolates DF+ACC DF+(NH4)SO4 RBR17 0.356 0.2954 RBR 19 0.8743 0.3875 RBR20 0.8530 0.5830 RBR25 0.8267 0.3742 RBR34 0.6743 0.4891 RBR40 0.8649 0.2965 RBR80 0.7492 0.3502 RBR83 0.9935 0.4683 RBR136 0.4162 0.2957 RBR144 0.998 0.5932 RBR155 0.8697 0.4700 RBR164 0.9985 0.3520 RBR165 0.7851 0.5831 LCRE8 0.7821 0.2843 RB1 0.9225 0.5932 3358 Int.J.Curr.Microbiol.App.Sci (2019) 8(2): 3350-3362 Fig.1 Phosphate solubilization Index (% PSI) of non rhizobial root endophytic Bacteria of chickpea Fig.2 Indole acetic acid production (with tryptophan) of root non rhizobial root endophytic bacteria of chickpea 3359